Apparatus for generating a temperature gradient and methods for using the gradient to characterize molecular interactions

ABSTRACT

A novel apparatus for generating temperature gradients is described. The apparatus includes a semiconductive wafer and electrical connectors attached to, preferably, one of the edges of the wafer. Methods for transferring the temperature gradients to strata are described. The temperature gradients on the strata can be used for analyses of molecules, particularly biological macromolecules. The present invention also includes improved methods for determining the thermal stability of binding complexes.

BACKGROUND OF THE INVENTION

[0001] This invention relates to an apparatus that generates a thermalgradient, particularly on a wafer. This invention also relates tomethods of using the thermal gradient in molecular interactions,particularly for characterizing interactions involving biologicalmacromolecules.

[0002] The stability and interactions of biological macromolecules aredetermined by a number of forces including, for example, ionic forces,van der Waals forces, and hydrogen bonds. Hydrogen bonds are known to befairly weak and heat-labile forces in biological macromolecules. Smallchanges in the environment, in particular the temperature of thebiological macromolecules, can alter the intramolecular and/orintermolecular hydrogen bonding of the macromolecules. Biologicalmacromolecules, thus, can be sensitive to even small fluctuations in theenvironment.

[0003] Hybridization between nucleic acid molecules requires successfulformation of hydrogen bonds between complementary nucleic acidmolecules. Because hybridization relies on weak, heat-labile hydrogenbonds, hybridization is an exquisitely temperature sensitive process.Small fluctuations in the temperature and/or in the sequence of thenucleic acids can affect hybridization between complementary nucleicacid molecules.

[0004] Currently, information derived from hybridizations conducted ondeoxyribonucleic acid (DNA) chips is stimulating advances in drugdevelopment, gene discovery, gene therapy, gene expression, geneticcounseling and plant biotechnology. A DNA chip is a rigid flat surface,typically glass or silicon, with short chains of related nucleic acidsspotted in rows and columns on it. As an example, hybridization betweena fluorescently labeled single stranded nucleic acid molecule andnucleic acid molecules at specific locations on the chip can be detectedand analyzed by computer-based instrumentation.

[0005] Among the technologies for creating DNA chips arephotolithography, “on-chip” synthesis, piezoelectric printing and directprinting. Chip dimensions, the number of sites of DNA deposition(sometimes termed “addresses”) per chip and the width of the DNA spotper “address” are dependent upon the technologies employed fordeposition. The most commonly used technologies presently produce spotswith diameter of 50-300 micrometers (μm). Photolithography producesspots that can have diameters as small as 1 μm. Technologies for makingsuch chips are described, for example, in U.S. Pat. No. 5,925,525 toFodor et al., U.S. Pat. No. 5,919,523 to Sundberg et al., U.S. Pat. No.5,837,832 to Chee et al. and U.S. Pat. No. 5,744,305 to Fodor et al.which are incorporated herein by reference.

[0006] Hybridization to nucleic acids on DNA chips can be monitored, forexample, by fluorescence optics, by radioisotope detection, and massspectrometry. The most widely-used method for detection of hybridizationemploys fluorescence-labeled DNA, and a computerized system featuring aconfocal fluorescence microscope (or an epifluorescence microscope), amovable microscope stage, and DNA detection software. Technicalcharacteristics of these microscope systems are described in U.S. Pat.No. 5,293,563 to Ohta, U.S. Pat. No. 5,459,325 to Hueton et al. and U.S.Pat. No. 5,552,928 to Furuhashi et al. which are incorporated herein byreference. Further descriptions of imaging fluorescently labeledimmobilized biomolecules and analysis of the images are set forth inU.S. Pat. No. 5,874,219 to Rava et al., U.S. Pat. No. 5,871,628 toDabiri et al., U.S. Pat. No. 5,834,758 to Trulson et al., U.S. Pat. No.5,631,734 to Stern et al., U.S. Pat. No. 5,578,832 to Trulson et al.,U.S. Pat. No. 5,552,322 to Nemoto et al. and U.S. Pat. No. 5,556,539 toMita et al. which are incorporated herein by reference.

[0007] Currently, manipulations performed with DNA chips are limited toprotocols in which all of the samples on a chip are at about the sametemperature. Simple and inexpensive methods of creating temperaturedifferentials on DNA chips would greatly expand the repertoire ofprocedures available that can be performed on a DNA chip.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the invention pertains to an apparatus. Theapparatus includes a semiconducting wafer and two electrical connectorsthat are adjacent to each other on the wafer. Each of the connectors areattached to the wafer at an attachment site on the wafer with a gapdisposed between the two attachment sites. A power source is connectedto the wafer through the two electrical connectors.

[0009] In a further aspect, the invention pertains to a method ofgenerating a temperature gradient. The method includes attaching twoelectrical connectors to a semiconducting wafer, wherein each of theconnectors are adjacent to each other and attached to the wafer at anattachment site with a gap disposed between the attachment sites. Themethod also includes connecting a power source to the wafer through thetwo electric connectors.

[0010] In another aspect, the invention pertains to a method ofanalyzing biological macromolecules. The method includes establishing atemperature gradient on a semiconducting wafer having a stratum disposedthereupon. The stratum has one or more samples that include biologicalmacromolecules in thermal contact with the temperature gradient. Thewafer has two electrical connectors connected to opposite poles of anelectrical power source. The method also includes evaluating the samplesto determine thermal stability of complexes formed with the biologicalmacromolecules in the samples. The samples are evaluated by measuring aproperty of the sample.

[0011] In a further aspect, the invention pertains to a method ofconducting nucleic acid hybridization. The method includes establishinga temperature gradient on a stratum disposed on a semiconducting wafer.One or more samples including nucleic acid molecules are disposed on thestratum that is in thermal contact with the temperature gradient. Twoelectrical connectors are connected to the wafer and to opposite polesof an electrical power source. The method also includes performing ahybridization protocol on the one or more samples to determinetemperature effect based on the gradient.

[0012] In yet another aspect, the invention pertains to a method ofassessing binding complex interactions. The method includes establishinga temperature gradient on a semiconducting wafer having a stratumdisposed thereupon. The stratum has one or more samples, each sampleincluding one or more members of a binding complex in thermal contactwith the temperature gradient. The wafer has two electrical connectorsconnected to opposite poles of an electrical power source. The methodalso includes evaluating the samples to determine thermal stability ofthe binding complex on the stratum.

[0013] In a further aspect, the invention pertains to a method ofgenerating a temperature gradient on a stratum. The method includesplacing the stratum in thermal contact on a surface having a temperaturegradient wherein the stratum has low thermal conductivity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic diagram of a thermal gradient apparatus.

[0015]FIG. 2 is a top view of the thermal gradient apparatus.

[0016]FIG. 3 is a side view of the thermal gradient apparatus of FIG. 2.

[0017]FIG. 4a is a top view of the wafer of FIG. 2 with three paralleltracks used for temperature measurement.

[0018]FIG. 4b is a plot of temperature versus position on the wafer foreach of the tracks indicated on the surface of the wafer in FIG. 4a.

[0019]FIG. 5a is a schematic illustration of an apparatus in which atemperature gradient is formed by thermal conduction alone.

[0020]FIG. 5b is a plot of temperature versus position for three tracksindicated in FIG. 5a using a silicon wafer in the apparatus.

[0021]FIG. 5c is a plot of temperature versus position for three tracksindicated in FIG. 5a using a glass microscope slide in the apparatus.

[0022]FIG. 6a is a top view of a thermal gradient apparatus with a glassslide on the wafer.

[0023]FIG. 6b is a plot of temperature versus position for each of thetracks indicated on the surface of the slide in FIG. 6a.

[0024]FIG. 6c-FIG. 6k are plots of temperature versus position for oneof the tracks on the surface of the slide in FIG. 6a when thetemperature controller of the apparatus was set to 40° C. 45° C., 50°C., 55° C., 60° C., 65° C., 70° C., 75° C. and 80° C., respectively.

[0025]FIG. 7a is a top view of a thermal gradient apparatus with threeglass slides on the wafer. Each slide has a drop of water that iscovered by a coverslip.

[0026]FIG. 7b is a plot of temperature versus position for each of thetracks shown in FIG. 7a.

[0027]FIG. 8a is a top view of a thermal gradient apparatus with afluidic cell on the wafer.

[0028]FIG. 8b is a detailed top view of the fluidic cell shown in FIG.8a.

[0029]FIG. 8c is a side view of the fluidic cell shown in FIG. 8b.

[0030]FIG. 8d is a plot of temperature versus position for each of thetracks shown in FIG. 8a.

[0031]FIG. 9a is a top view of a thermal gradient apparatus with anacrylamide gel formed between two glass slides and placed on the wafer.

[0032]FIG. 9b is a side view of the gel assembly shown in FIG. 9a.

[0033]FIG. 9c is a plot of temperature versus position for each of thetracks on the surface of the slide shown in FIG. 9a.

[0034]FIG. 10a is a diagram of a chip containing a single row ofimmobilized DNA spots exposed to a 40-70° C. gradient duringhybridization with a complementary labeled nucleic acid probe.

[0035]FIG. 10b is a hypothetical result of the experiment depicted inFIG. 10a.

[0036]FIG. 11a is a diagram of a chip containing three different DNAmolecules, each immobilized in a different row, exposed to a temperaturegradient during hybridization with a labeled nucleic acid probe.

[0037]FIG. 11b is a hypothetical result of the experiment depicted inFIG. 11a.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] It has been discovered that stable temperature gradients can begenerated on an electrically semiconductive wafer connected to a currentsource. Temperature gradients are generated on the wafer, in a gradientapparatus, by applying a voltage to two physically separated sites onthe wafer. The gradients are stable and approximately linear oversignificant distances.

[0039] It has also been discovered that a temperature gradient can betransferred onto or into an adjacent stratum when the stratum is placedin thermal contact with a surface having the temperature gradient, evenif the stratum has low thermal conductivity. These temperature gradientscan be used in efficient approaches for the evaluation of properties ofbiological macromolecules.

[0040] The gradient apparatus includes an electrically semiconductivewafer and two electrical connectors that are attached to the wafer atadjacent sites, which are referred to as the attachment sites. When thetwo electric connectors are connected to the opposite poles of a powersource, temperature gradients can be produced on the wafer thatgenerally are substantially perpendicular to an attachment line that isderived by connecting the attachment sites. In other words, thetemperature at a given site on the wafer depends on the distance fromthe attachment line.

[0041] The wafer can include a proximal edge and a distal edge. Theproximal edge of the wafer can include the attachment sites or can beclose to the attachment sites. The side opposite the proximal edge isreferred to as the distal edge of the wafer. The attachment line isderived by connecting the respective edges of the two attachment sitesclosest to the distal edge of the wafer. A gradient is formedapproximately perpendicular to the attachment line. Away from theconnectors, the temperature is approximately independent ofdisplacements along lines parallel to the attachment line. Thus,separate tracks can be defined perpendicular to the attachment linehaving approximately the same temperature gradient.

[0042] Control circuitry controlling the flow of current between thewafer and the power source can provide adjustable but stable andreproducible temperature gradients. The control circuitry can include,for example, a temperature controller, a temperature sensor, a relayswitch, a transformer and the like.

[0043] Small incremental changes in the temperature can be generated andadvantageously detected on the surface of the wafer. The thermalgradients on the wafer generally are generated by resistive heating ofthe wafer and thermal conduction of the heat. In preferred embodiments,reproducible, stable temperature gradient increments of between about0.1° C./mm and about 1.0° C./mm are generated on the semiconductivewafer. Applications of the stable temperature gradients include, but arenot limited to, determination of the thermal stabilities of duplexnucleic acid molecules, polypeptide:polypeptide complexes,polypeptide:ligand complexes, polypeptide:nucleic acid complexes,polypeptide:lipid complexes, polypeptide:carbohydrate complexes and thelike. As applied to nucleic acids, the ability to detect thermalsensitivity differences can provide diagnostic tools for use in avariety of applications.

[0044] The temperature gradients generated on surfaces such as asemiconductive wafer can be surprisingly transmitted to one or morestratum placed on the wafer. The stratum can include, for example, DNAchips, protein chips, glass microscopic slides, fluidic cells, liquids,acrylamide gels and the like. When the strata are placed on a wafer in agradient apparatus of the invention, a thermal gradient can also bedetected on the strata.

[0045] Analyses on a variety of biological/chemical molecules can beconducted by using the temperature gradients generated on strata. Inparticular, thermal stability determinations can be made using thetemperature gradients on a stratum. The molecules can be linked onto orinto the stratum. The molecules are generally immobilized on thestratum. By “immobilized”, it is meant that the molecules are notsubstantially removed or substantially repositioned during subsequentwashing or other experimental manipulations. Molecules can beimmobilized to the stratum, for example, by covalent bonding,hydrophobic bonding, ionic bonding and absorption.

[0046] The biological/chemical molecules can include biologicalmacromolecules, for example, nucleic acid molecules, polypeptides,carbohydrates and the like. The biological/chemical molecules can alsoinclude, for example, drugs, lipids, hormones, ligands and the like,which may or may not be macromolecules.

[0047] Cells and tissues, for example, may also be immobilized onmicroscopic glass slides by chemically fixing the cells onto the glassslide. Chemical fixatives can include, for example, formalin, ethanol,formaldehyde, paraformaldehyde, glutaraldehyde and the like.

[0048] The use of a gradient apparatus of the present invention with DNAchips or protein chips and various labeled probes has many diagnosticapplications. In one embodiment, temperature-dependent hybridizationsbetween single-stranded nucleic acid molecules immobilized on a chip anda labeled nucleic acid probe can be performed to form a nucleic acidduplex. Nucleic acid molecules at different positions on the chip areexposed to different temperatures based on their location relative tothe temperature gradient. The hybridization signal can be correlatedwith the temperature. The results of these studies can establish therelatedness of similar nucleic acid molecules by estimating the numberof base mismatches between each of nucleic acid molecule samples and thelabeled nucleic acid probe. The gradient apparatus can be used toadvantageously identify even single-base mismatches in a nucleic acidduplex.

[0049] In another embodiment, temperature dependent analyses of bindingcomplexes can be performed on chips. The complexes can be, for example,antigen:antibody complexes, enzyme:substrate complexes, receptor:ligandcomplexes, polypeptide:polypeptide complexes, polypeptide:lipidcomplexes, polypeptide:carbohydrate complexes, polypeptide: nucleic acidcomplexes and the like. Binding complexes at different positions on thechip can be exposed to different temperatures based on their locationrelative to the temperature gradient. The results of these experimentscan establish the relative dissociation strengths of related bindingcomplexes.

[0050] A. Apparatus

[0051] The gradient apparatus of the present invention includes anelectrically semiconductive wafer, a pair of electrical connectorsattached to the wafer and a power source. The electrical connectors areadjacent to each other, but physically separate, when connected to thewafer. The gradient apparatus may also include control circuitry toobtain a desired temperature gradient.

[0052] The semiconductive wafer can be made from a variety of materialsincluding, for example, germanium, silicon, gray (crystalline) tin,selenium, tellurium and boron. The semiconductive wafer is preferablymade from silicon and germanium and more preferably from silicon. Thesemiconductive wafer preferably has a substantially uniform surfacecomposition. The electrical conductivity of semiconductive wafers canbe, for example between about 10⁴ and about 10⁻³ ohm⁻¹cm⁻¹. Preferably,the electrical conductivity of semiconductive wafers is between about10⁴ and about 10⁻² ohm⁻¹cm⁻¹.

[0053] The semiconductive wafer can be doped. Doping can increase ordecrease the conductivity of the wafer. Techniques for doping are knownin the art and are described, for example, in Campbell, S. A. TheScience and Engineering of Microelectronic Fabrication; New York, OxfordUniversity Press; 1996; pp. 98-102, incorporated herein by reference.Doping agents can include, for example, boron, phosphorous, arsenic andthe like. Preferable semiconductive wafers are boron doped siliconwafers. Doping of a silicon wafer, for example, can change theelectrical conductivity of the silicon wafer from 10⁻¹ ohm⁻¹cm⁻¹ toabout 10³ ohm⁻¹cm⁻¹.

[0054] The semiconductive wafer is preferably substantially flat andlevel to retain a stratum placed on the wafer for an indefinite periodof time. The wafer, preferably, is also smooth to optimize efficientconductive heat transfer to the stratum.

[0055] In some preferred embodiments, the wafer can include a pluralityof edges, preferably four edges. The wafer may be in the shape of apolygon, for example, a pentagon, a hexagon and the like. The wafer mayalso be curvilinear. The wafer can be substantially shaped, for example,as a rectangle, a square or other parallelograms. The wafer edges mayhave corresponding substantially rectangular edges, square edges and thelike. The corners, for example, may be 90° corners, clipped corners andthe like. Alternatively, the wafer may have rounded corners and thus,can include, for example, ovoid wafers, rectangular-like wafer withrounded corners. Wafers with rounded corners may not have corners thatclearly delineate the different edges. In these embodiments, a rectanglewith corresponding edges can be drawn over the edge of the wafer toapproximate the shape of the wafer. This approximate rectangle can beused to describe the gradient and relative points on the wafer surface.All of these wafers, however, can be used in the gradient apparatusdescribed herein.

[0056] Two electrical connectors are generally attached to the waferadjacent to each other but physically separated. By adjacent, it ismeant that the two electrical connectors are approximately at the sameedge but are not in physical contact with each other. Preferably, theelectrical connectors are attached to the same edge of the wafer,relative to an approximating rectangle, if relevant. As described above,the line derived from connecting the attachment site edges, of eachattachment site, closest to the distal end of wafer is the attachmentline.

[0057] Generally, a gap is disposed between the attachment sites of theelectrical connectors. The distance between the attachment sites of theelectrical connectors, i.e. the gap, is preferably between about 2 mmand about 180 mm, and more preferably between about 5 mm and about 50mm. The electrical connectors can be edge connectors, for example, cardedge connectors.

[0058] The electrical connectors can be connected to the opposite polesof a power source through electrically conductive wires. The powersource can be an alternating current power source or a direct currentpower source. Preferably, the power source has voltage of between about2 volts and about 40 volts and more preferably between about 4 volts and24 volts.

[0059] The temperature gradients on the surface of the wafer aregenerally substantially perpendicular to the attachment line that can bedefined as the x-axis. The temperature gradients are, thus, generatedalong a y-axis, perpendicular to the x-axis (attachment line). In otherwords, the temperature changes according to the distance from theattachment line. The temperature is approximately constant at equaldistances from the attachment line.

[0060] Generally, the temperature is highest near the attachment lineand progressively decreases with distance away from the attachment line.Preferably, the decrease in the temperature along any y-axis isapproximately linear. Preferably, movement along the x direction for anyvalue of y does not result in any substantial temperature change.

[0061] The temperature along the x direction at or near the attachmentline may vary slightly. The temperatures along a line parallel to and atabout 20 mm from the attachment line preferably are about the same. Morepreferably, the temperatures along a line parallel to and about 15 mmfrom the attachment line are about the same. Even more preferably, thetemperatures along a line parallel to and about 10 mm from theattachment line are about the same.

[0062] The gradient apparatus preferably includes control circuitry. Thecontrol circuitry of the gradient apparatus can include, for example, atemperature sensor, a temperature controller, a relay switch and atransformer. The temperature sensor is generally physically attached tothe wafer and electrically connected to the temperature controller. Therelay switch and the transformer can be operatively connected betweenthe power source and the wafer. Feedback control between the temperaturesensor and the temperature controller is used to open or close the relayswitch, thereby regulating power to the wafer and controlling itstemperature.

[0063] The temperature sensor is generally attached to the wafer surfacein the gradient apparatus. The temperature sensor is preferablypositioned in the gap between the two electrical connectors of thegradient apparatus.

[0064] The temperature sensor can detect the temperature of the waferand is associated with the upper surface of the wafer. Preferably, thetemperature sensor is a resistive temperature sensor, a thermocouple, athermodiode, a thermotransistor, thermoresistor or thermistor. Morepreferably, the temperature sensor is a 100 ohm platinum resistivetemperature detector (RTD). Temperature controllers can be purchasedfrom a number of commercial sources. A suitable temperature controllerincludes, for example, temperature controller model 982 from WatlowEngineering, Winona, Minn.

[0065] The temperature controller and the temperature sensor may beparts of separately fabricated electrical circuits. Alternatively, theymay comprise a single integrated circuit.

[0066] In some embodiments, the gradient apparatus may operate withoutthe feedback control provided by the temperature controller and sensor.The gradient can then be dependent on the power source, ambienttemperature and the like.

[0067] A variety of suitable relay switches can be used in the gradientapparatus. Suitable relay switches include, for example, a solid staterelay switch, a reed relay switch and the like. Preferably, the relayswitch is a solid state relay switch.

[0068] A transformer can be used to change the voltage from a powersource, for example, an alternating current power source, to the wafer.The transformer generally is in electrical series with the power sourceand the relay switch, for example, as illustrated in FIG. 1.

[0069] The apparatus may optionally be mounted in a walled structurethat supports the wafer in a substantially flat manner and houses thecontroller. The housing may be fabricated from various suitablematerials including plastic and/or metal. Preferably, the housing isplastic, such as polypropylene or polycarbonate so that the housing maybe molded in an inexpensive fashion.

[0070] The apparatus may also include a support, preferably molded fromelectrically insulating materials such as silicone rubber, underneaththe distal edge of the wafer.

[0071] The apparatus of the invention may also include a commercialpersonal computer, work station or a self-contained microprocessor. Inone embodiment, the computer receives temperature information from thetemperature sensor and executes software commands that cause thecontroller to open or close the relay, thereby regulating the poweravailable to heat the wafer.

[0072] In another embodiment, the computer may be electrically attachedby transmission cables to a relay controller and to an electronicsensing device (an analog to digital converter) that is electricallyconnected to the temperature sensor. In this embodiment, the computerreceives temperature information from the analog to digital converterand executes software commands to the relay controller.

[0073] FIGS. 1-3 show an illustrated embodiment of a gradient apparatus.A schematic of gradient apparatus 100 is shown in FIG. 1. Gradientapparatus 100 includes silicon wafer 110, electrical connectors 114 aand 114 b and electrically conductive wires 116 a and 116 b.Electrically conductive wires 116 a and 116 b can be connected to powersource 126 through control circuitry.

[0074] Control circuitry can include a number of components. In theembodiment illustrated, control circuitry includes transformer 120,temperature sensor 130, temperature controller 136 and relay switch 140.Temperature sensor 130 is positioned in gap 134 between connectors 114 aand 114 b. Temperature sensor 130 and transformer 120 are connected to atemperature controller 136 such as the model 982 available from WatlowEngineering, Winona, Minn. A solid state relay switch 140 is connectedby electrical wire 116 b in one electrical series to the controller 136.

[0075] A top view of gradient apparatus 100 is shown in FIG. 2. Gradientapparatus 100 includes silicon wafer 110 and two electrical connectors114 a and 114 b attached to wafer 110 at the proximal edge. The distaledge of wafer 110 is supported by wafer gasket 166. Posts 168 supportthe wafer gasket 166 and the electrical connectors 114 a and 114 b.

[0076]FIG. 3 is a side view of apparatus 100 shown in FIG. 2. The wafergasket 166 and housing 160 support the distal edge of wafer 110 (the endopposite the electrical connectors 114 a and 114 b).

[0077] B. Generation of Temperature Gradients

[0078] The gradient apparatus of the present invention can producestable temperature gradients on a wafer when the electrical connectorsof the apparatus are connected to a power source. The temperaturegradient increments generated on the wafer can be small andreproducible.

[0079] The gradient apparatus can be used to establish many differentgradients. In order to generate a desired temperature gradient, thetemperature controller of the apparatus can be set to a desired setpoint temperature. The temperature on the wafer is generally highestnear the attachment line and decreases away from the attachment line.The actual gradient generated on the wafer can depend on, for example,the set point temperature selected, the placement of the temperaturesensor on the wafer, the electrical resistance of the card connectors,ambient temperature and also on the composition of the wafer.

[0080] The temperature detected at the attachment line is the upperlimit of the temperature gradient on the wafer and is preferably withinabout 20° C., and more preferably, within about 10° C. of the set pointtemperature.

[0081] The temperature range of the gradients generated on the wafer canbe manipulated by adjusting the set point temperature of the temperaturecontroller in the apparatus of the invention. The temperature controllercan be set, for example, at about 75° C. to obtain a temperaturegradient of between about 75° C. and about 50° C. Similarly, thetemperature controller can be set, for example, at about 45° C. toobtain a temperature gradient of between about 45° C. and 20° C.

[0082] Preferably, the apparatus generates stable temperature gradientincrements of less than about 1° C. per millimeter (mm). Morepreferably, the apparatus generates stable temperature gradientincrements of between about 0.1° C./mm and 0.5° C./mm. The ability togenerate such small temperature gradient increments can be applied tomany different analyses of biological/chemical molecules.

[0083] The temperature gradient on the wafer can be determined bymeasuring the temperature at various locations along a y-axis. A plot oftemperature versus position, i.e. distance from the x-axis or attachmentline, can produce a temperature gradient profile. The slope of thetemperature gradient profile also can provide the temperature gradientincrements generated on the wafer.

[0084] The slope of the temperature gradient profile for a given waferand for given conditions is generally substantially reproducible. Thegradient apparatus can be calibrated for a wafer and fixed by settingthe current. Once calibrated, reproducible temperature gradients aregenerated by setting the set point of the controller.

[0085] The slope of the temperature gradient profile, thus theincrements, may be decreased by additionally attaching a heating sourceto the wafer at the distal edge of the wafer. The presence of theconnectors as described above and an additional heating source at thedistal edge may result in a temperature gradient profile with a smallerslope.

[0086] The slope of the temperature gradient profile may be increased byattaching a cooling source at the distal end from the connectors. Thecooling can be performed, for example, by using a fan to sweep ambienttemperature across the end opposite the electrical connectors.Preferably, the cooling can be performed by thermoelectric coolingprovided by a peltier device attached to the wafer, an anodized aluminumheat sink attached to the wafer at the distal end and the like.

[0087] The temperature gradient generated on the surface of the wafercan be monitored in a non-obtrusive manner with an infrared-sensitivecamera system, image acquisition software, data logging software, amemory card for storing the acquired data and data plotting software.Other methods for monitoring can include methods of gradient analysisinvolving placement of temperature sensitive electronic circuits such asresistance temperature devices in direct contact with the wafer. Thedetectors, however, can act as heat sinks and distort the gradientprofile.

[0088] The gradient apparatus of the present invention can generate thedesired temperature gradients using resistive heating and thermalconduction. In one embodiment, as shown in FIG. 4a and FIG. 4b, with anambient temperature of about 20° C., a temperature gradient of about0.30° C./mm was generated on the surface of a boron doped silicon wafer.In addition, the plot in FIG. 4b, indicates that the gradient generatedcan be monotone and substantially linear. In temperature gradientprofiles described herein, the distances are measured from theattachment line, i.e. 0 mm is the attachment line.

[0089] For comparison, an apparatus for producing a temperature gradientby thermal conduction alone (no resistive heating) is illustrated inFIG. 5a. The thermal conduction apparatus of FIG. 5a includes surface182 that can be either a silicon wafer or a glass microscope slideheated at one end with a 20 ohm resistor. As shown in FIG. 5b, thetemperature gradient when the surface is of a silicon wafer is about 10°C./mm. The method of the invention described herein, thus, can produce atemperature gradient that is approximately 30 times shallower than thegradient produced by thermal conduction alone on a wafer.

[0090] As shown in FIG. 5c, the gradient produced when the surface is ofa microscope slide can be approximately 3.7° C./mm using thermalconduction alone. The methods described herein, thus, can produce atemperature gradient that is approximately 10 times shallower than thegradient produced by conduction alone on a glass microscope slide.

[0091] C. Application of Temperature Gradients

[0092] The temperature gradients generated on the semiconductive waferusing the gradient apparatus described herein can be transferred to oneor more strata placed on the wafer. The thermal gradients on the stratacan, in turn, be used to assess the thermal stabilities of moleculesincluding, for example, nucleic acids, polypeptides, carbohydrates,lipids, drugs, ligands, combinations thereof and the like. The thermalstability of complexes of the chemical/biological molecules and theirrespective binding partner(s) can be determined using the methodsdescribed herein.

[0093] The molecules to be analyzed are generally placed on a stratumsuch as a glass microscope slide, a silicon chip, an acrylamide gel,nitrocellulose, a charged nylon membrane and the like. By placing thestratum containing the molecules on the wafer of the invention,performing biological manipulations, and then removing the stratum fromthe wafer, the wafer can be reused many times. When biologicalmanipulations are carried out, as shown in the examples below, thetemperature gradient on the wafer is transferred to the stratum.

[0094] Samples that can be analyzed in the gradient apparatus caninclude any number of chemical/biological molecules. Samples can includeisolated or purified macromolecule preparations such as isolated nucleicacids and polypeptides. Samples can also include drugs, hormones, andthe like. In addition, samples can include tissues, parts of tissues,partially purified tissue extractions, cell preparations, living cellsand other biological material.

[0095] Suitable strata can include, for example, glass chips,microscopic glass slides, acrylamide gels, fluidic cells, liquids,coverslips for the glass slides and the like. Strata can be any otherstrata that are employed in medical diagnostics, molecular biology andcellular biology at temperatures, preferably ranging from ambienttemperature to about 100° C. The thermal conductivity of the strata canvary. Surprisingly, the temperature gradient can be transmitted tostrata that have low thermal conductivity. The temperature gradient canbe transferred to glass, for example, that has a thermal conductivitybetween about 0.1-1.0 watts/meter/° K.

[0096] Stratum can include one component such as a DNA chip or a proteinchip. The stratum can also include a plurality of components, forexample, a fluidic cell having a base, a glass slide, liquid and acover, a slide assembly having two slides with acrylamide gel disposedbetween the slide, and the like.

[0097] In particular, a temperature gradient on a wafer of the inventioncan be transferred to the surface of a glass or silicon DNA chip in thegradient apparatus. Thus, any samples that may be present on the DNAchip would be subjected to the corresponding temperature of the DNA chipat the particular location.

[0098] In one illustrated embodiment, glass microscopic slide stratum190 is placed on wafer 110 of the invention as shown in FIG. 6a. Thetemperature gradients along the y-axes of the slide were collected andanalyzed by an infrared imaging system and then plotted in FIG. 6b.These results demonstrate that y-axes on microscopic glass slide 190have temperature gradients of approximately 0.3° C./mm.

[0099] The temperature gradients along y-axes of the slide when thecontroller's set point was adjusted to 40° C., 45° C., 50° C., 55° C.,60° C., 65° C., 70° C., 75° C. and 80° C., respectively are plotted inFIG. 6c-FIG. 6k. These results demonstrate that the invention canproduce gradients with slopes between 0.1° C./mm and about 0.5° C./mm.The slope of the gradient is determined by the setpoint. Thus, setpointsof 40° C., 70° C. and 80° C. generate, respectively, gradients withslopes of 0.1° C./mm, 0.3° C./mm, and 0.5° C./mm.

[0100] In another illustrated embodiment shown in FIG. 7a, apparatus 100includes three glass microscope slides 190 placed on silicon wafer 110.A small drop of water, approximately 50 microliters in volume, can beplaced onto the center of each of the three microscopic slides. Eachdrop of water can then be covered with a glass microscope slidecoverslip 196. The three slide assemblies can then be placed on wafer110 with a preformed temperature gradient and analyzed by thermalimaging after one minute. The plotted results of the apparatus in FIG.7a are shown in FIG. 7b. The results in FIG. 7b indicate that the y-axeson coverslips 196 atop each of the slides 190 can have temperaturegradients of approximately 0.30° C./mm.

[0101] In another illustrated embodiment shown in FIG. 8a, apparatus 100with a DNA chip fluidic cell 210 containing glass microscopic slide 190and liquid film with a volume of approximately 120 microliters is showndisposed on wafer 110. FIG. 8b illustrates a more detailed top view ofthe fluidic cell 210 shown in FIG. 8a. Assembled fluidic cell 210 caninclude base 214, preferably made from lucite, with a machined recess218 that can accommodate microscopic slide 190, a machined groove fittedwith an o-ring 220, lucite lid 240 with two filling holes 224 andmachine screws 230 that tighten the lid against o-ring 220. In use, aglass microscopic slide with DNA is placed in the cell, the lid isplaced on the gasket and the machine screws are used to tighten the lidagainst the gasket. The tightened cell can be water-tight, and has anairspace, approximately 0.1 mm high between the surface of the slide andthe lid, with a volume of approximately 120 microliters. Fluid can beintroduced through one of the fill holes and both fills can then besealed. The assembled cell with a fluid film atop the slide is placed onthe wafer of the invention with a preformed temperature gradient and thetemperature along the lucite lid of the cell is collected and analyzedby an infrared imaging system and then plotted. A plot of thetemperature gradients on the lid of cell is shown in FIG. 8d andindicates that the y-axes of the lid of the cell can have temperaturegradients of approximately 0.3° C./mm.

[0102] The temperature gradients can also be transmitted to and throughan acrylamide gel disposed between two glass slides. Two glass slideswith acrylamide gel between them can be placed on the wafer. In FIG. 9aand FIG. 9b, two glass slides 190 a and 190 b are disposed on the waferwith an acrylamide gel between glass slides 190 a and 190 b. The lineartracks, shown in FIG. 9a, are the tracks on the upper surface of topglass slide 190 b. The temperature on the upper surface of the top glassslide 190 b can be measured using an infrared camera as described above.The upper surface of the top glass slide 190 b can have a temperaturegradient as shown in FIG. 9c. The presence of temperature gradients onthe top glass slide 190 b can indicate that the strata underneath theglass slide 190 b have approximately similar temperature gradients.Thus, the bottom glass slide 190 a and the acrylamide gel between theglass slides 190 a and 190 b can have approximately similar temperaturegradients as the top glass slide 190 b. The samples in the acrylamidegel can, thus, be exposed to the temperature gradients.

[0103] Samples can be placed on the stratum in order to assess thermalstability at a variety of temperatures. Generally, a plurality ofsamples are placed on the stratum at different locations on the stratum.As described above, samples that are placed at differing positions alonga y-axis perpendicular to the attachment line, i.e. samples havingdifferent y-coordinates, are generally exposed to differenttemperatures. Samples that are placed at the same y-coordinates but atdiffering points along an x-axis, i.e. samples have differingx-coordinates but same y-coordinates, are generally exposed to about thesame temperature.

[0104] The samples may be placed on the stratum using a variety oftechniques known in the art. A number of nucleic acid samples, forexample, may be immobilized on DNA chips as described in U.S. Pat. No.5,925,525 to Fodor et al., U.S. Pat. No. 5,919,523 to Sundberg et al.,U.S. Pat. No. 5,837,832 to Chee et al. and U.S. Pat. No. 5,744,305 toFodor et al. which are incorporated herein by reference. Nucleic acidmolecules, for example, can also be directly synthesized on the DNA chipby solid phase synthesis on derivatized chips. The samples may also beimmobilized onto the stratum by chemical crosslinking using crosslinkingagents, ultraviolet crosslinking and the like. Crosslinking agents andmethods of crosslinking are known in the art.

[0105] The sample placed on the stratum can include a single type ofmolecule such as a preparation of a single stranded nucleic acidmolecule, a preparation of an antigen and the like. Potential bindingpartners of these molecules, for example, the complementary strand ofthe single stranded nucleic acid, the antibody of a antigen:antibodycomplex can be subsequently added to the stratum and allowed to bind tothe immobilized sample on the stratum. A labeled probe can be added tothe stratum that directly or indirectly binds to the moleculesimmobilized on the stratum. In some embodiments, the labeled probe isthe binding partner of the immobilized molecule.

[0106] The labeled probe can provide a means for detecting the thermalstability of the molecules at the specific location on the stratum.Detection of the thermal stability can be performed by observing,locating and/or quantifying the label that is specifically associatedwith the sample on the stratum. The signal generated for detection ofthermal stability can be derived directly or indirectly from theinteraction between the sample and the labeled probe. Detection and/ormeasurement of the signal of the labeled probe can then be indicative ofand/or correlated with the thermal stability of the molecules on thestratum.

[0107] The label of the labeled probe can be for example, a radioactiveatom, a fluorophore, a chromophore, a chemiluminescent reagent, anenzyme capable of generating a colored, fluorescent or chemiluminescentproduct, or a binding moiety capable of reaction with another moleculeor particle which directly carries or catalytically generates thesignal. Binding moieties can be attached to the probe and include, forexample, biotin that binds tightly to streptavidin or avidin anddigoxygenin that is tightly bound by anti-digoxygenin antibodies. Theavidin, streptavidin and antibodies, in turn, are easily attached tochromophores, fluorophores, radioactive atoms, and enzymes capable ofgenerating colored, fluorescent or chemiluminescent signals.

[0108] Detection of the labeled probe, bound directly or indirectly tothe molecules of the sample, can include detecting fluorescence,chemiluminescence, radiolabels and the like by one or more ofmicroscopy, photography, autoradiography and fluorimetry. Detection canalso be measured, for example, by measuring the visible or ultravioletabsorbance.

[0109] In some embodiments, the sample can include a binding complex,i.e. two members of a binding complex, such as a double stranded nucleicacid molecule, a receptor:ligand complex and the like. The bindingcomplex can be immobilized onto the stratum. When exposed to atemperature gradient, the binding complex, if dissociated, may exhibit ameasurable property. For example, a binding complex may have ameasurable fluorescence that changes upon dissociation.

[0110] The chips generally include a plurality of samples. All of thesamples on the chip may be the same. Alternatively, each track of thechip may contain different samples. Within each track, the samples arepreferably equivalent so that the equivalent samples can be exposed todifferent temperature. Track, as referred to herein, is a row of samplesalong one of the y-axis, i.e. the samples in a track have the samex-coordinate and different y-coordinates.

[0111] In the apparatus of the invention, a pair of electricalconnectors are attached to the wafer as described above, and connectedto a power source. In preferred embodiments, the temperature controllerof the gradient apparatus is set to a desired temperature determined bythe nature of the samples to be analyzed.

[0112] The samples on the chip are exposed to different temperaturesthat can be directly related to position on the stratum due to thegradient profile of the wafer which is efficiently transferred to thestratum. Generally, the slope of the temperature gradient on the stratumis approximately similar to the slope of the temperature gradient on thewafer.

[0113] The interaction between the labeled probe and the sample orsamples at various temperatures can be determined since the samples atdifferent positions on the stratum are exposed to differenttemperatures. Analysis of the information generated by these type ofstudies can identify the thermal dissociation temperature of thespecific complexes formed; that is, the temperature at which the membersof the binding complex no longer remain stably associated with eachother because of thermal instability associated with, for instance,temperature-dependent changes in molecular shape, ortemperature-dependent rupture of hydrogen bonds.

[0114] In some embodiments, for example, a detectable signal, generatedby the labeled probe and indicative of a nucleic acid hybridizationevent, may be present for samples, at locations specifying temperaturesof 60° C. and 60.3° C., but not at 60.6° C. The hybridization betweenthe samples and the labeled probe, thus, is stable at temperatures up toabout 60.3° C., but is not stable at temperatures above about 60.6° C.

[0115] The apparatus of the invention can be used to determine thethermal stability of nucleic acid duplexes by conducting nucleic acidhybridizations on a stratum on the wafer. Methods for nucleic acidhybridization and thermal dissociation are known in the art. As appliedto DNA chips with the present invention, they would involve thefollowing sequential steps: obtaining or creating a DNA chip containingthe desired array of single stranded nucleic acid molecules immobilizedon its surface; obtaining or creating a suitable labeled nucleic acidprobe; annealing the labeled probe to the immobilized nucleic acids ofthe DNA chip disposed on the wafer having a temperature gradient;removing the unhybridized probe by repeating washing; and detecting theremaining hybridized probe. The labeled probe can be, for example, afluorescently labeled, short single stranded DNA molecule between about12 nucleotides long and about 20 nucleotides long. The results of suchanalyses would provide a graphic representation of thetemperature-dependence of nucleic acid hybridization.

[0116] The present invention can include methods of determining thestabilities of nucleic acid primers used in polymerase chain reaction(PCR). PCR is a method of amplifying nucleic acid target sequences. PCRis commonly used, and is described, for example, in PCR, A PracticalApproach; M. J. McPherson, P. Quirke, and G. R. Taylor, eds. IRL Press,Oxford, UK, 1991.

[0117] PCR is a technique involving multiple temperature cycles thatresult in the geometric amplification of specific polynucleotidespresent in a test sample, i.e. target sequences, each time a cycle iscompleted. One of the steps in this amplification of target sequences isthe hybridization of a single stranded oligonucleotide referred to as aprimer to a region close to or within the target sequence. Primerspreferably are deoxyribonucleotides. The primers can be between about 12and about 50 nucleotides in length and contain base sequences withWatson-Crick complementarity to sequences on one strand of the targetsequence.

[0118] The primers anneal to the target sequence in order for theamplification to occur. The temperature at which the primer is allowedto anneal with the target sequence is referred to as the annealingtemperature. A desirable annealing temperature is generally high enoughto suppress annealing at non-specific sites but low enough to allow forduplex formation between the complementary primer:target sequences. Atannealing temperatures that are too low, the primer may anneal atnon-specific sites. Thus, it is desirable to identify the highesttemperature at which primer:target sequence duplexes can be maintainedin solution.

[0119] In one embodiment of the present invention, the dissociationtemperature of specific PCR target:primer duplexes can be determined.Target DNAs can be placed, for example, in rows along the y-axis of thestratum. The stratum can then be placed in a fluidic cell, exposed to atemperature gradient, incubated with labeled primers, and washed. Theresulting pattern of labeling can identify the thermal stability of theprimer:target duplex. FIG. 10a and FIG. 10b illustrate an example ofthis embodiment and a hypothetical result. A DNA chip 270 with targetDNA arrayed in a row along the y-axis is shown in FIG. 10a. The targetDNA on the chip is exposed to a gradient of between about 40° C. andabout 70° C., and hybridized with a fluorescent primer. After washing toremove unbound primer, the distribution of label along the y-axis isdetermined. One hypothetical result is illustrated in FIG. 10b. Theresult in FIG. 10b shows that a labeled probe is present at a positionin the stratum corresponding to about 55° C., but is not present at aposition corresponding to about 55.6° C. The optimum temperature forthis primer:target combination, thus, is about 55.0° C.

[0120] In another embodiment, the temperature gradient on a wafer can beused to identify one or more mismatches between related nucleic acidmolecules. In particular, the thermal stability information obtainedfrom performing nucleic acid hybridizations in the gradient apparatuscan be used to identify the percentage of mismatch between two nucleicacid molecules. FIG. 11a illustrates one method of using the gradientapparatus for determining thermal stabilities of closely related nucleicacid molecules. Three samples of DNA differing by one base can beimmobilized on a chip and analyzed. Each of the samples can be placed ona different track with aliquots of the same sample placed at differentpositions within the track. A labeled oligonucleotide probe that isexactly complementary to one of the three DNA samples and covering thearea of the base mismatch(s) in the other two DNA samples can be addedto DNA samples on the chip. The data derived from such a study canresult in data, for example, as shown in FIG. 11b. Using this method asingle mismatch between the labeled probe and the DNA sample can bedetected.

[0121] In FIG. 11a and FIG. 11b, DNA chip 280 has three different DNAmolecules 17 a, 17 b, and 17 c arrayed in parallel rows. Molecule 17 bdiffers from molecule 17 c by one base change, and molecule 17 a differsfrom molecule 17 c by two base changes. This DNA chip can be placed in afluidic cell, hybridized to a labeled probe exactly complementary tomolecule 17 c and washed to remove unbound labeled probe. One possibleresult is the distribution of labeled probe along the y-axis for eachsample as shown in FIG. 11b. The highest stability is seen between DNAmolecule 17 c and the probe, followed by DNA molecule 17 b. The probeand DNA molecule 17 a form the least stable complex. This result isconsistent with the fact that there are no mismatches between DNAmolecule 17 c and the probe and that there is one mismatch between DNAmolecule 17 b and the probe. This result is also consistent with thefact that there are two mismatches between DNA molecule 17 a and theprobe. The present invention, thus, can be used to identify single basedifferences between DNA molecules.

[0122] The thermal stabilities of various binding complexes can also beevaluated using the gradient apparatus of the present invention. Thebinding complexes may include, but are not limited to,polypeptide:nucleic acid complexes, nucleic acid complexes,polypeptide:polypeptide complexes such as antigen:antibody complexes,polypeptide:carbohydrate complexes, polypeptide:lipid complexes,polypeptide:hormone complexes, receptor:drug complexes and the like. Inparticular, the thermal stability of antigen:antibody, enzyme:substrate,and receptor:ligand complexes can be established.

[0123] One member of the binding complex can be immobilized on thestratum. The second member of the binding complex is preferably added tothe stratum and allowed to bind to the immobilized member to form abinding complex. One of the members of the binding complex may include alabel, preferably the second member. Alternatively, a labeled probe maybe added that interacts with only the binding complex and is indicativeof the presence of a complex.

[0124] In some embodiments, binding complexes may be immobilized. Thedetection methods can include signals indicative of either the presenceof the binding complex or dissociation of the binding complex.

[0125] In one embodiment, monoclonal antibodies directed againstdifferent epitopes of a single antigen can be immobilized in rows alongy-axes on a protein chip. Using a temperature gradient, for example,between about 20° C. and about 45° C. and a labeled antigen probe,binding stabilities can be determined using procedures similar to theprocedures described for DNA chips above. Briefly, the labeled antigenprobe can be added and allowed sufficient time to bind to the monoclonalantibodies on the chip. Unbound labeled antigen can be washed away. Thesites with bound labeled antigen can be identified using any of theprotocols described above and correlated with the temperature at thesite. The results can provide thermal stability information related tothe various antigen:antibody complexes on the chip.

[0126] The strength of interaction between a polypeptide receptor andspecific hormones, drugs, and/or other ligands also can be analyzed.Natural hormones, for example, and their synthetic analogs can bechemically linked in rows parallel to the y-axis of a stratum. Using atemperature gradient, for example, between about 20° C. and about 45°C., the labeled receptor probe, and the procedures described above, thethermal stabilities of receptor:hormone complexes can be determined.

[0127] Candidate drugs for therapeutic use can be chemically linked inrows parallel to the y-axis of a stratum. Using a temperature gradient,for example, between about 20° C. and about 45° C., labeled drugreceptor probe, and the procedures described above, the thermalstabilities of individual receptor:drug complexes can be assessed.

[0128] The discussion described herein relates to some applications ofthe temperature gradients generated on a semiconductive wafer. The useof thermal gradients is not limited to only the applications describedherein. Other applications of using the thermal gradients are alsocontemplated by the inventors and will be apparent to those skilled inthe art.

EXAMPLES Example 1 Generation of a Temperature Gradient on a Wafer

[0129] This example illustrates the generation of a temperature gradienton a silicon wafer by resistive heating and thermal conductivity. Thisexample also compares the gradients of the invention with a temperaturegradient formed by thermal conductivity alone.

[0130] Temperature gradient formed by resistive heating and thermalconductivity gradient was formed with an apparatus shown in FIGS. 1-3. A114 mm×114 mm×0.6 mm boron doped silicon wafer was diced from a circular150 mm wafer purchased from WaferNet (San Jose, Calif.). Card edgeconnectors were connected to the wafer and to the power source. —Thetemperature controller (model 982; Watlow Engineering; Winona, Minn.),was set to about 75° C. The temperature controller determinedtemperature with the electrical signal received from a temperaturesensor that was a 100 ohm platinum RTD (Minco; Fridley, Minn.). Thetemperature controller/temperature sensor maintained the temperature atabout 75° C. by the technique of proportional integrationdifferentiation (PID) looping known in the art. The temperaturedetermined by the controller was within 1.0° C. of the set point withinfive minutes of operation, and stayed at 75° C.±0.5° C. indefinitely.After the set point had been attained, the temperature profile of thewafer's surface was taken with an IR SNAPSHOT digital camera (InfraredSolutions: Plymouth, Minn.).

[0131]FIG. 4a shows three parallel tracks on the surface of the wafer,perpendicular to the attachment line drawn between the connectors of theinvention, each separated by approximately 5 mm. The temperature versusposition plot along these tracks, shown in FIG. 4b, has three lines thatwere approximately linear, with slopes of approximately 0.3° C./mm.

[0132] A thermal conductivity gradient was formed with an apparatuscomparable to the apparatus depicted in FIG. 5a. A 20 ohm resistor wasglued to one end of a 25.4 mm×76.2 mm×0.6 mm silicon wafer. The resistorreceived 10 volts of alternating current from a variable-powertransformer, and thereby provided heat to one end of the wafer. One hourafter power was provided to the resistor, the temperature profile of thesurface of the wafer was determined as described above.

[0133]FIG. 5a showed three parallel tracks on the surface of the wafer,perpendicular to the resistor, each separated by about 5 mm. Thetemperature versus position plot along these tracks, shown in FIG. 5b,has three approximately exponential curves, each with a slope ofapproximately 10° C./mm.

[0134] Comparison of the plots in FIG. 4b and FIG. 5b demonstrated thatthe gradient apparatus of the invention (resistive heating with thermalconduction) produced a shallow linear gradient extending at least 100 mmfrom the source of power, while thermal conductivity alone produced asteep exponential gradient that essentially ends within 10 mm of thesource of power.

Example 2 Generation of a Gradient on a Glass Slide

[0135] This example compares the gradient formed on the surface of aglass slide by resistive heating and thermal conductivity to thegradient formed by conductivity alone.

[0136] The temperature gradient formed by resistive heating and thermalconductivity was generated as described in example 1. A glass slidehaving dimensions of about 25 mm×75 mm×1 mm was placed on the wafer asindicated in FIG. 6a. The temperature gradient was visualized asdescribed in example 1.

[0137]FIG. 6a shows three parallel tracks on the surface of the glassslide, each separated by about 5 mm. The temperature versus positionplot along these tracks, shown in FIG. 6b, has three lines that wereapproximately linear, with slopes of approximately 0.3° C./mm.

[0138] A gradient with thermal conductivity alone was formed with theapparatus described in example 1 and shown in FIG. 5a. A 20 ohm resistorwas glued to one end of a 25.4 mm×76.2 mm×1.0 mm glass slide. Theresistor received 10 Volts of alternating current from a variable-powertransformer, and thereby provided heat to one end of the slide. One hourafter power was provided to the resistor, the temperature profile of thesurface of the wafer was determined as described in example 1.

[0139]FIG. 5a shows three parallel tracks on the surface of the glassslide, each separated by about 5 mm. The temperature versus positionplot along these tracks, shown in FIG. 5c, produced three exponentialcurves, each with slopes of approximately 3.7° C./mm.

[0140] Comparison of the plots in FIG. 6b and FIG. 5c demonstrated thatthe apparatus of FIG. 6a produced a shallow linear gradient extendingacross the entire microscope slide, while thermal conductivity aloneproduced a steep exponential gradient that essentially ended within 20mm of the source of power.

Example 3 Generation of Different Gradients on a Glass Slide

[0141] This example demonstrates generation of different gradient rangeson a glass slide.

[0142] The temperature gradient produced by resistive heating andthermal conductivity was generated as described in example 2, and thetemperature gradient was visualized as described in example 1. To obtaindifferent temperature gradients, the temperature controller was set to40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80°C., respectively.

[0143]FIG. 6a shows three parallel tracks on the surface of the glassslide, each separated by about 5 mm. The temperature versus positionplot along track 1 at each of the set point temperatures is shown inFIG. 6c-FIG. 6k. These results demonstrate that the invention canproduce gradients with slopes between 0.1° C./mm and 0.5° C./mm. Theslope of the gradient is determined by the set point temperature. Thus,set points of 40° C., 70° C., and 80° C. generate, respectively,gradients with slopes of 0.1° C./mm, 0.3° C./mm and 0.5° C./mm.

Example 4 Gradient on a Glass Microscope Coverslip

[0144] This example illustrates the generation of temperature gradientson the surface of glass coverslips placed on a glass microscopic slidewith a drop of water. The assembly with the glass slide and thecoverslips were placed on a wafer having a temperature gradient.

[0145] The gradient apparatus as shown in FIG. 7a was used and thetemperature gradient was visualized as described in example 1. A drop ofwater was placed on top of 3 glass slides. A glass microscope coverslipwas placed on each drop of water. Each coverslip had dimensions of about22 mm×50 mm×0.1 mm. FIG. 7a shows the apparatus with the microscopicglass slides and the coverslips. FIG. 7a also shows a parallel track oneach coverslip that was analyzed as described in Example 1. FIG. 7b is aplot of temperature versus position of the three parallel tracks shownin FIG. 7a. As shown in FIG. 7b, a temperature gradient can be formedand maintained on the surface of the coverslip. The temperature gradientformed on the surface of the coverslip was about 0.3° C./mm.

Example 5 Generation of a Gradient on the Surface of a Fluidic Cell

[0146] This example illustrates the generation of a gradient on thesurface of a fluidic cell containing a glass microscopic slide with DNA.

[0147] The resistive heating and thermal conductivity gradient wasgenerated as in example 1. The fluidic cell illustrated in FIG. 8b waspositioned on the wafer of the invention as illustrated in FIG. 8a.Temperature on the surface of the top plastic cover of the fluidic cellwas visualized as described in example 1.

[0148]FIG. 8a shows three parallel tracks on the surface of the fluidiccell, each separated by 5 mm. The temperature versus position plot alongthese tracks, shown in FIG. 8c, produced three lines that wereessentially linear, with slopes of approximately 0.3° C./mm. Therefore,the temperature gradient of the invention can be transferredsuccessively through the lucite base of the fluidic cell, a glass slide,the fluid film covering the glass slide and the lucite lid of thefluidic cell.

Example 6 Generation of a Gradient Along the Length of an Acrylamide Gel

[0149] This example illustrates the generation along the length of anacrylamide gel with a gradient apparatus of the present invention.

[0150] The resistive heating and thermal conductivity gradient wasgenerated as in example 1. A 5% acrylamide gel was formed between twoglass slides as illustrated in FIG. 9b and placed on the wafer of theinvention as illustrated in FIG. 9a. Temperature on the surface of thetop glass cover of the slide was visualized as described in example 1.

[0151]FIG. 9a shows three parallel tracks on the surface of the upperglass slide of the acrylamide gel, each separated by about 5 mm. Thetemperature versus position plot along these tracks, shown in FIG. 9c,produced three lines that were essentially linear, with slopes of about0.3° C./mm. Therefore, the temperature gradient of the invention can betransferred successively through a glass slide, an acrylamide gel andanother glass slide.

[0152] Although the present invention has been described with referenceto preferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. An apparatus comprising: a semiconducting wafer;two electrical connectors adjacent to each other on the wafer, each ofthe connectors attached to the wafer at an attachment site with a gapdisposed between the two attachment sites; and a power source connectedto the wafer through the two electrical connectors.
 2. The apparatus ofclaim 1 wherein the semiconducting wafer has a substantially rectangularshape with corresponding rectangular edges.
 3. The apparatus of claim 2wherein both attachment sites are near one of the edges.
 4. Theapparatus of claim 1 wherein the semiconducting wafer comprises asilicon wafer.
 5. The apparatus of claim 4 wherein the semiconductingwafer comprises a doping agent.
 6. The apparatus of claim 5 wherein thedoping agent is selected from the group consisting of boron, phosphorousand arsenic.
 7. The apparatus of claim 1 wherein the electric connectorsand the power source are connected by electrical wires.
 8. The apparatusof claim 7 further comprising control circuitry between the wafer andthe power source.
 9. The apparatus of claim 8 wherein the controlcircuitry comprises a temperature sensor disposed in the gap andelectrically connected to a temperature controller.
 10. The apparatus ofclaim 9 further comprising feedback control between the temperaturesensor and the temperature controller to maintain the measurement of thetemperature sensor within a selected range, and wherein the feedbackcontrol opens or closes a relay switch.
 11. The apparatus of claim 8wherein the control circuitry further comprises an electricaltransformer connected in series between the power source and theelectrical connectors.
 12. The apparatus of claim 1 further comprisingone or more stratum disposed on the wafer.
 13. The apparatus of claim 12wherein the stratum are selected from a group consisting of a DNA chip,a protein chip, a fluidic cell, a microscopic slide, liquid, coverslip,acrylamide gel and combinations thereof.
 14. The apparatus of claim 12further comprising samples disposed on the stratum.
 15. The apparatus ofclaim 14 wherein the samples comprise molecules selected from the groupconsisting of nucleic acid molecules, polypeptides, carbohydrates,lipids, hormones, drugs and combinations thereof.
 16. The apparatus ofclaim 14 further comprising labeled probes disposed on the stratum. 17.The apparatus of claim 16 wherein the labeled probes are selected fromthe group consisting of fluorescent labeled probes, chemiluminescentlabeled probes and radiolabeled probes.
 18. The apparatus of claim 12further comprising members of a binding complex disposed on the stratum.19. The apparatus of claim 1 wherein a temperature gradient formed onthe wafer is perpendicular to an attachment line derived from connectingthe two attachment sites.
 20. The apparatus of claim 1 wherein the waferhas clipped corners.
 21. The apparatus of claim 1 wherein the twoattachment sites are separated by a distance of between about 2 mm andabout 180 mm.
 22. The apparatus of claim 1 wherein the wafer comprises asubstantially uniform composition.
 23. A method of generating atemperature gradient comprising: attaching two electrical connectors toa semiconducting wafer, wherein each of the connectors are adjacent toeach other and attached to the wafer at an attachment site with a gapdisposed between the attachment sites; and connecting a power source tothe wafer through the two electric connectors.
 24. The method of claim23 wherein the apparatus further comprises a temperature sensor attachedat the same edge of the wafer as the electrical connectors andelectrically connected to a temperature controller.
 25. The method ofclaim 24 further comprising selecting a set point temperature on thetemperature controller.
 26. The method of claim 23 wherein thetemperature gradient formed is substantially perpendicular to anattachment line connecting the two connectors.
 27. The method of claim26 wherein the temperature gradient formed is between about 0.1° C. permillimeter and about 1.0° C. per millimeter.
 28. The method of claim 26wherein the temperature gradient is between about 0.25° C. permillimeter and about 0.7° C. per millimeter.
 29. The method of claim 23wherein the wafer comprises silicon.
 30. The method of claim 23 furthercomprising placing one or more stratum on the wafer to generate atemperature gradient on the stratum.
 31. The method of claim 30 whereinthe stratum comprise low thermal conductivity materials.
 32. The methodof claim 30 wherein the stratum are selected from the group consistingof microscopic glass slides, fluidic cells, liquid, cover slips,acrylamide gel, DNA chips, protein chips and combinations thereof.
 33. Amethod of analyzing biological macromolecules comprising: establishing atemperature gradient on a semiconducting wafer having a stratum disposedthereupon, the stratum having one or more samples comprising biologicalmacromolecules in thermal contact with the temperature gradient, thewafer having two electrical connectors connected to opposite poles of anelectrical power source; and evaluating the samples to determine thermalstability of complexes formed with the biological macromolecules in thesamples wherein the samples are evaluated by measuring a property of thesample.
 34. The method of claim 33 wherein the temperature gradient issubstantially perpendicular to an attachment line derived fromconnecting the two electrical connectors.
 35. The method of claim 33wherein the biological macromolecules are nucleic acids.
 36. The methodof claim 35 wherein the stratum comprises a DNA chip with the nucleicacids.
 37. The method of claim 35 wherein the stratum comprises anacrylamide gel having the nucleic acids.
 38. The method of claim 35wherein the evaluating comprises characterizing the thermal stabilitiesof nucleic acid molecules.
 39. The method of claim 35 wherein theevaluating comprises characterizing the thermal stability of a complexformed by two single stranded nucleic acid molecules.
 40. The method ofclaim 39 wherein the evaluating comprises characterizing the thermalstability of a complex formed by two single stranded nucleic acidmolecules having one or more base mismatches.
 41. The method of claim 36wherein the evaluating comprises adding a labeled probe to the DNA chip,washing unbound labeled probe, detecting the activity of the labeledprobe at various positions on the DNA chip and determining the thermalstability of the interaction between the labeled probe and the nucleicacid molecules on the chip by correlating the activity of the labeledprobe with the temperature of the sample at the various positions on theDNA chip.
 42. The method of claim 41 wherein the probe is a labelednucleic acid.
 43. The method of claim 42 wherein the correlatingidentifies the percentage of mismatch between the labeled nucleic acidprobe and the nucleic acid molecules of the DNA chip.
 44. The method ofclaim 35 wherein the evaluating comprises characterizing the thermalstabilities of nucleic acid hybrids formed with primers for use inpolymerase chain reaction protocols.
 45. The method of claim 33 whereinthe biological macromolecules are polypeptides.
 46. The method of claim45 wherein the stratum is a glass chip.
 47. The method of claim 45wherein the polypeptides are selected from the group consisting ofantigens, antibodies, enzymes, receptors and fragments thereof.
 48. Themethod of claim 45 wherein the temperature gradient on the stratum isbetween about 20° C and about 45° C.
 49. The method of claim 33 theevaluating comprises adding a labeled probe to the stratum.
 50. Themethod of claim 49 wherein the evaluating comprises detecting theactivity of the labeled probe at various positions on the temperaturegradient and determining the stability of the interaction between thelabeled probe and the biological macromolecule by correlating theactivity of the labeled probe with the position on the temperaturegradient.
 51. The method of claim 49 wherein the label of the labeledprobe is selected from the group consisting of fluorescent label, aradioactive label and a chemiluminescent label.
 52. A method ofconducting nucleic acid hybridization comprising: establishing atemperature gradient on a stratum disposed on a semiconducting wafer,wherein one or more samples comprising nucleic acid molecules aredisposed on the stratum that is in thermal contact with the temperaturegradient, two electrical connectors being connected to the wafer and toopposite poles of an electrical power source; and performing ahybridization protocol on the one or more samples to determinetemperature effect based on the gradient.
 53. The method of claim 52wherein the stratum is selected from the group consisting of DNA chipsand microscopic slides.
 54. The method of claim 52 wherein thehybridization protocol comprises adding a labeled probe on the stratum.55. The method of claim 52 wherein the method further comprisesidentifying the one or more samples that hybridized with the labeledprobe.
 56. The method of claim 52 wherein the hybridization protocolcomprises conditions that can identify one or more base mismatchesbetween the labeled probe and the unlabeled nucleic acid molecule. 57.The method of claim 52 wherein the wafer is substantially rectangularand comprises a plurality of edges, the two electrical connectorsconnected to the wafer at the same edge.
 58. The method of claim 52wherein the temperature gradient is substantially perpendicular to aline segment derived from connecting attachment sites of the twoelectrical connectors.
 59. The method of claim 52 wherein the stratumcomprises one or more equivalent samples in a row parallel to thetemperature gradient.
 60. A method of assessing binding complexinteractions comprising: establishing a temperature gradient on asemiconducting wafer having a stratum disposed thereupon, the stratumhaving one or more samples, each sample comprising one or more membersof a binding complex in thermal contact with the temperature gradient,the wafer having two electrical connectors connected to opposite polesof an electrical power source; and evaluating the samples to determinethermal stability of the binding complex on the stratum.
 61. The methodof claim 60 wherein the evaluating comprises adding a labeled probe tothe stratum.
 62. The method of claim 61 wherein one of the members ofthe binding complex comprises the labeled probe.
 63. The method of claim62 further comprising detecting the activity of the labeled member ofthe binding complex at various positions on the temperature gradient anddetermining the thermal stability of the binding complex by correlatingthe activity of the labeled member with the position on the temperaturegradient.
 64. The method of claim 60 wherein the binding complexcomprises a nucleic acid duplex.
 65. The method of claim 60 wherein thebinding complex comprises two or more polypeptides.
 66. The method ofclaim 60 wherein the binding complex comprises a nucleicacid:polypeptide complex.
 67. The method of claim 60 wherein the bindingcomplex comprises a nucleic acid:drug complex.
 68. The method of claim60 wherein the binding complex comprises an antigen:antibody complex.69. The method of claim 60 wherein the binding complex comprises areceptor:drug complex.
 70. The method of claim 60 wherein the bindingcomplex comprises a lipid:polypeptide complex.
 71. The method of claim60 wherein the binding complex comprises carbohydrate:polypeptidecomplex.
 72. The method of claim 60 wherein the binding complexcomprises a biological macromolecule and a binding partner of thebiological macromolecule.
 73. A method of generating a temperaturegradient on a stratum comprising placing the stratum in thermal contacton a surface having a temperature gradient, the stratum having lowthermal conductivity.
 74. The method of claim 73 wherein the surface isa silicon wafer.
 75. The method of claim 73 wherein the surfacecomprises aluminum blocks.
 76. The method of claim 73 wherein thetemperature gradient on the surface is generated by thermoelectricPeltier devices.
 77. The method of claim 73 wherein the stratumcomprises materials selected from the group consisting of glass, siliconand plastic.